Microtubule plus end tracking proteins or +TIPS have been implicated in numerous fundamental cellular processes. For example, the yeast complex of Myo2p, a type V myosin, Bim1p, the yeast homolog of the mammalian +TIP EB1, and Kar9p, which links Myo2p to Bim1p, facilitates mitotic spindle orientation. In mouse melanocytes, myosin Va is recruited on to the surface of melanosomes by a receptor complex containing Rab27a present in the melanosome membrane and melanophilin, which links myosin Va to Rab27a. We now find that melanophilin is also a +TIP. Moreover, myosin Va tracks the microtubule plus end in a melanophilin-dependent manner. Data showing that over expression and siRNA knockdown of EB1 have opposite effects on melanophilin: microtubule interaction, that melanophilin interacts directly with EB1, and that deletion from melanophilin of a region similar to one in APC involved in EB1 binding blocks melanophilin?s ability to plus end track, argue that melanophilin tracks the plus end indirectly by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells have retained in the form of a myosin Va-melanophilin-EB1 complex the connection between the microtubule plus end and actin seen in yeast in the form of the Myo2p-Kar9p-Bim1p complex. Given melanophilin?s documented role in coupling Rab27a-bearing melanosomes to myosin Va, we suggest that the complex of myosin Va-melanophilin-EB1 at the microtubule plus end may serve to focus the transfer of melanosomes from microtubules to actin at this location. Acanthamoeba CARMIL was previously shown to co-purify extensively with capping protein (CP) and to bind pure CP with an affinity of ~ 0.4 iM. We now find that this interaction serves to sequester CP in an inactive state. Even more strikingly, we show that CARMIL uncaps actin filaments previously capped with CP. While full length (FL) CARMIL (residues 1-1121) possesses these activities, C-terminal fragments like GST-P (residues 940-1121) that contain CARMIL?s CP binding site are at least ten times more active. We localized the full activities of GST-P to its C-terminal 51 residues (51aa; 1071-1121). The sequestering and uncapping activities of 51aa are potent: 20 and 100 nM 51aa, when mixed with 3 nM CP prior to the addition of seeds, blocked CP function by 50% and 100%, respectively, and the addition of 30 nM 51aa to actin filaments previously capped with 3 nM CP restored the rate of actin assembly to that of the CP-free control within seconds. Sequence alignments identified a stretch of 25 residues within 51aa that are highly conserved among protozoan, fly, worm, and vertebrate CARMIL proteins (CARMIL Homology domain-3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-capping activity of GST-AP (residues 862-1121). Finally, we found that GST-AP binds CP ~20 fold more tightly than does FL-CARMIL (apparent Kd ~20 nM). This observation, together with the elevated anti-capping protein activities of C-terminal fragments relative to FL-CARMIL, suggests that FL-CARMIL might exist primarily in an auto-inhibited state. Consistent with this, proteolytic cleavage of FL-CARMIL with thrombin generated an ~14-kDa C-terminal fragment that expresses full sequestering and uncapping activities. We propose that, following some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist by sequestering free CP in an inactive form and by actively uncapping capped actin filaments. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly.